1. Field of the Invention
The invention is a method of enzymatic synthesis of isotopically labeled metabolites. In particular, the invention relates to a rapid and convenient synthesis of isotopically labeled carbohydrates, citric acid cycle intermediates, amino acids and ribose mononucleotides, all of which may be derived from labeled pyruvate, lactate or L-alanine.
2. Description of Related Art
Methods of preparing sugars and nucleotides labeled in the carbohydrate moiety have traditionally utilized the reactions of classic organic chemistry, (1-3). Typically, an aldose is prepared labeled at the anomeric carbon (C-1) by condensing an aldose one carbon shorter with isotopically labeled cyanide for the preparation of carbon-13 or carbon-14 labeled sugars. Deuterium, tritium or oxygen labeled isotopes may be prepared by subsequent reduction with hydrogen isotope in the presence of isotopically enriched aqueous solution, (4-6). Using this reaction sequence, a mixture of C-2 epimers is obtained, which then must be separated by chromatography. Yields of the desired product are usually in the range of 40-80%. Labeling at C-2 may be achieved by a molybdate ion catalyzed epimerization followed by chromatography. In order to label carbon sites other than C-1 or C-2, the entire sequence of reactions must be repeated, using the appropriately labeled material during one of the turns of the cycle. Not unexpectedly, each turn of such a cycle severely diminishes the yield of the desired product.
Labeling at internal carbon sites of a carbohydrate is best accomplished by enzymatic routes. Due to the permissive nature of rabbit muscle aldolase to accept a range of three to six carbon aldehydes, a variety of labeled sugar phosphates can be prepared enzymatically if the appropriately labeled substrate is provided (7-13). The labeled substrate is prepared by a chemical method, such as those described above, or enzymatically prepared from glycerol. In general, synthesis of these precursors may require several steps.
An improved method of isotopically labeled carbohydrate synthesis using enzymes of the glycolytic pathway provides good yields of hexose and pentose sugars and the various phosphorylated intermediates leading to these sugars (14,15). This method is essentially a single step reaction requiring mixing labeled pyruvate with enzymes of the glycolytic and neoglycolytic pathway and allowing the reaction to proceed to completion. Although this method provides a simple and rapid synthesis, high levels of ATP and NADH are required to force the reaction to completion and the final product requires a time-consuming chromatographic separation.
No general chemical procedure for the isotopic labeling of L-amino acids exists. Methods have been described for preparing [2,3-.sup.13 C] DL-alanine (16), [1-.sup.13 C] DL-alanine (17-19), [2-.sup.13 C] DL-alanine (20) and for the asymmetric synthesis of unlabeled L-alanine (21). The latter method, however, is difficult to adapt to an isotopically enriched synthetic scheme because of the difficulty in preparing the labeled starting material, .alpha.-acetamidoacrylic acid. Methods used for preparing [1-.sup.13 C] DL-alanine have also been used to prepare racemic mixtures of other amino acids labeled at the carbonyl carbons. Methods also exist for the isotopic labeling of aspartic and glutamic acids at their side chain carboxyls (22-23). More recently, methods have become available for isolating amino acids from bacteria grown on .sup.13 C-labeled substrates. Selection of mutant strains allows the possibility of site specific labeling patterns (24).
The above classical chemical methods suffer because they only allow for a few simple labeling patterns and because a racemic mixture of products is obtained, ultimately decreasing the overall yield of the desired enantiomer. The bacterial method, while of greater overall utility, requires knowledge of the isolation of amino acids from hydrolyzed proteins.
Of the citric acid cycle intermediates, [2, 3, 2-.sup.13 C.sub.2 ] succinate has been synthesized by classical methods from diethylmalonate and 2-.sup.13 C-ethylchloroacetate while [1,4-.sup.13 C.sub.2 ] and [2,3-.sup.13 C.sub.2 ] succinate have been prepared from .sup.13 C-enriched potassium cyanide and/or [1,2-.sup.13 C]1,2-dibromoethane (25). Using the first of these two chemical methods, the product can be labeled at any chosen position. However, the difficulty in obtaining .sup.13 C-labeled malonic acid may make the method less than desirable. The second of the two methods is useful only for labeling of the two carboxyl carbona or the two internal carbons.
[1-.sup.13 C] or [4-.sup.13 C] malate can be prepared from the appropriately labeled aspartic acid using chemical methods (25). Similarly the reaction has been carried out enzymatically using an aspartate transaminase catalyzed reaction (26). Both methods have the disadvantage of requiring the appropriately labeled aspartic acid substrate.
[5-.sup.13 C, .sup.18 O] citrate has been prepared enzymatically from [1-.sup.13 C] acetate and unlabeled oxaloacetate (27). No convenient chemical synthesis of isotopically labeled citrate exists.
Isotopically labeled products are of increasing value in biochemical and medical research. Already analysis of metabolic disposition by nuclear magnetic resonance techniques has been used to monitor normal and pathological metabolism (28-30). Deuterated and perhaps .sup.13 C isoptopically labeled compounds may also have clinical applications in magnetic resonance imaging.
With these ever-increasing medical and clinical applications, there is a need for adequate and reliable supplies of labeled compounds particularly suited for safe use in humans. With attention turning to new techniques capable of detecting labeled metabolites and intermediates associated with metabolic processes, there is increased pressure to develop rapid and efficient methods of obtaining labeled compounds suitable for metabolic diagnosis, including citric acid cycle intermediates, carbohydrates and nucleotides. Methods which do not require time consuming isolation procedures, involve simple purification and are adaptable in providing a wide selection of labeled compounds would alleviate some of the problems associated with presently available methods of obtaining these labeled compounds.